Abstract

Ca2+ is a ubiquitous intracellular signalling molecule that is involved in the regulation of numerous cellular functions. To date Ca2+ influx pathways present in white fat adipocytes have not been characterised. Additionally impaired [Ca2+]i management is implicated in the induction of the insulin resistant state in adipocytes. As adipocytes have a prominent role in the management of energy homeostasis, the presence of Ca2+ influx pathways was examined.

Initial [Ca2+]i measurements confirmed the presence of functional Ca2+ influx and efflux pathways in adipocytes. Further [Ca2+]i measurements identified the Cav1.3 Voltage-gated Ca2+ channel (VGCC). The presence of the α1 subunit of Cav1.3 channel protein in adipocytes was confirmed by Western blotting, the expression of which was reduced in adipocyte samples sourced from Zucker obese rats.

Initial [Ca2+]i imaging experiments utilising conditions of elevated extracellular K+ (50mM) did not stimulate Ca2+ influx. The plasma membrane potential (Vm) regulates many physiological processes, including cellular Ca2+ influx by VGCCs, with dysregulations in Vm underlying functional pathologies. K+ is widely believed to be the predominant ion that controls Vm for many cell types, however, whether K+ regulates adipocyte Vm is also unknown, prompting, investigation into the ionic species involved in the regulation of Vm in primary and differentiated 3T3-L1 adipocytes. As insulin and β-adrenoceptors regulate adipocyte function, their effect on Vm was also explored.

The Vm of primary and 3T3-L1 adipocytes were -34.14mV (n=68) and -28.5mV (n=88) respectively. Elevation of extracellular K+ from 5.6mM to 50mM had no significant effect on the Vm of either type of adipocyte. The role of Cl- on adipocyte Vm was then investigated. Reduction of extracellular Cl- from 138 to 5mM, by equimolar substitution with Gluconate significantly depolarised the Vm of both primary and 3T3-L1 adipocytes. Patch clamp investigations also revealed a role of Na+ in adipocyte Vm. Neither insulin (100nM) or the β-adrenocpetor agonist isoprenaline (10µM) significantly changed adipocyte Vm.

The role of Cl- in adipocyte Vm is indicative of the presence of Cl- channels, however electrophysiological studies failed to characterise the Cl- currents underlying adipocyte Vm.

Overall, further investigations are required to characterise not only the Ca2+ influx pathways in adipocytes, and the roles thereof, but also the means by which they are regulated.